The present invention is concerned with an immunomodulatory substance.
It is more particularly concerned with an immunostimulating factor capable of activating human neutrophil leukocytes, hereinafter designated neutrophil-activating factor (NAF).
The neutrophil leukocytes (neutrophils) are the most common leukocytes and account for about ⅔ of the white cells in human blood. They have one main function, which is to protect the host organism against microbial infections. The neutrophils are mobile, are responsive to chemotactic stimuli generated upon infection, and are able to move into infected tissues to kill the microorganisms. The killing depends on the ability of the neutrophils to engulf the microorganisms and to release oxygen radicals and microbiocidal enzymes (B. M. Babior, New Engl. J. Med. 298 [1978] 659-668 and M. Baggiolini, Experientia 40 [1984 ] 906-909). The release of such products depends on activation of the neutrophils. Substances like the neutrophil-activating factor described herein could thus be employed to enhance neutrophil activation and therefore the antimicrobial defense mechanism of the human body.
We have now found that human monocytes secrete a factor that induces exocytosis and the respiratory burst (oxygen radical formation and enzyme release) in human neutrophils and thus displays neutrophil-activating properties which are important in antimicrobial activity.
The factor can be obtained from the culture fluid of stimulated human monocytes and has an apparent molecular weight of approximately 6000, more precisely about 6500, in SDS-gel electrophoresis.
Monocytes are phagocytic cells similar to neutrophils. In contrast to neutrophils, however, monocytes are long-lived. They migrate from the blood into all possible tissue sites where they transform into macrophages. The transformation from monocytes to macrophages can also be observed in cell culture experiments. The macrophages in the tissues have a variety of functions, mainly phagocytosis of unwanted material and production of a great variety of peptides and proteins which are secreted. Macrophages collect at the site of persisting infection and it is at these locations that the production of NAF by these cells could enhance the host-defence capabilities of neutrophils and thus have a pathophysiologically relevant function.
NAF is characterized by its neutrophil-activating properties, i.e. the induction of the so-called respiratory burst with production of oxygen radicals and the induction of enzyme release. In molecular terms NAF, is characterized by the following properties: an apparent molecular weight of about 6500 in SDS-gel electrophoresis, an isoelectric point of approximately 8.6, resistance to heat up to 80xc2x0 C. and to a number of denaturing agents but susceptibility to proteases, suggesting that NAF is a polypeptide. The action of NAF on human neutrophils is similar to that of two known chemotactic stimuli, the anaphylatoxin C5a and the bacterial peptide N-formyl-L-methionyl-L-leucyl-L-phenyl-alanine (fMLP), but is mediated by a surface receptor to which NAF binds and which differs from the receptors of any known agonist of human neutrophils. NAF is produced in culture by human monocytes but not by human lymphocytes. Production depends on the presence of a stimulus like bacterial lipopolysaccharide (LPS), phytohaemagglutinin (PHA), or concanavalin A (ConA) and on stimulus concentration and incubation time. It is inhibited by cycloheximide, indicating that de novo synthesis of protein is involved.
As already indicated, monocytes and macrophages are abundant sources of a variety of bioactive peptides and proteins (C. F. Nathan, J. Clin. Invest. 79 [1987] 319-326). They have been identified as the producers of three distinct cytokines: interleukin l (IL-l), tumor necrosis factor (TNF) and interferon-alpha (IFN-xcex1). A number of reports have been published showing that monocytes and macrophages also produce factors acting on neutrophils which are different from those mentioned above. Since they are active on neutrophils these factors are presented hereafter in some detail. It was reported in various publications that alveolar macrophages release factors which are chemotactic for neutrophils (J. A. Kazmierowski et al., J. Clin. Invest. 59 [1977] 273-281; W. W. Merrill et al., J. Clin. Invest. 65 [1980] 268-270 and G. W. Hunninghake et al., J. Clin. Invest. 66 [1980] 473-483) and which can enhance the antimicrobial defense in the lung. It was shown subsequently that these factors enhance the microbiocidal activity of neutrophils (J. E. Pennington et al., J. Infect. Dis. 148 [1983] 101-109 and J. Clin. Invest. 75 [1985] 1230-1237). Purification by gel filtration and chromatofocusing led to the identification of a protease-sensitive factor (termed NAF) produced by alveolar macrophages, with a molecular weight of 6000 and an isoelectric point of 7.6. This factor was reported to be weakly chemotactic and to enhance the killing of phagocytosed bacteria by neutrophils without, however, inducing by itself the production of oxygen radicals nor the release of enzymes. A similar mechanism of enhanced anti-microbial activity was described by other workers (A. Ferrante et al., Clin. Exp. Immunol. 56 [1984] 559-566), who showed that human neutrophils required the addition of culture media from monocytes or macrophages to induce the killing of Neigleria fowleri. Granulocyte-activating mediators (GRAM) produced by LPS-simulated human monocytes were described by other laboratories (A. Kapp et al., J. Invest. Dermatol. 86 [1986] 523-528 and F. E. Maly et al., Lymphokine Res. 5 [1986] 21-33). Two GRAM species were described, a major one with an apparent molecular weight of 60,000 and a minor one with an apparent molecular weight of 10,000, which induced a delayed respiratory burst response in human neutrophils, as revealed by chemiluminescence. These factors are sensitive to heat and trypsin and their production is dependent on stimulation of the monocytes with LPS and, apparently, on de novo protein synthesis.
Several reports describe factors derived from monocytes and/or macrophages with chemotactic activity towards neutrophils. A factor termed xe2x80x9cmononuclear cell-derived chemotaxinxe2x80x9d (MOC), apparently a peptide with molecular weight of 10,000 which differs from GRAM was reported, (E. Kownatzki et al., Clin. Exp. Immunol. 64 [1986] 214-222). A neutrophil chemotactic factor produced by human blood monocytes stimulated with LPS, with a molecular weight of approximately 10,000 and an isoelectric point of 8-8.5 was also known (T. Yoshimura et al., J. Immunol. 139 [1987] 788-793). Finally it was also reported that rat peritoneal macrophages stimulated in culture with LPS release a selective neutrophil chemotactic factor (F. Q. Cunha et al., J. Med. Biol. Res. 19 [1986] 775-777 and Eur. J. Pharmacol. 129 [1986] 65-76).
Due to the preliminary nature of the biochemical information contained in these reports it is impossible to speculate about structural similarities and differences among the various factors described.
Subsequently to the priority date(s) which is (are) being claimed for the present invention, purification of a peptide probably corresponding to NAF was described by van Damme et al. (J. Van Damme et al., J. Exp. Med. 167 [1988] 1364-1376) and a sequence identical to that of NAF was reported by Gregory et al., (H. Gregory et al., Biochem. Biophys. Res. Commun. 151 [1988] 893-890) for a peptide that was purified from supernatants of lectin-stimulated human lymphocytes. The cDNA coding for MDNCF (T. Yoshimura et al., Proc. Nat. Acad. Sci. USA 84 [1987] 9233-9237) was recently cloned (K. Matsushima et al., J. Exp. Med. 167 [1988] 1883-1893) and found to correspond to the 3-lOC cDNA (J. Schmid and C. Weissmann, J. Immunol. 139 [1987] 250-256). Schmid and Weissmann have shown that the peptide encoded by the 3-10C cDNA has structural homology with platelet factor 4, beta-thromboglobulin, connective tissue-activating peptide III (CTAP-III) and interferon-gamma-inducible peptide (gamma-IP-10). These molecules are also homologous to a 73-residue peptide (MGSA) with melanoma growth-stimulating properties (A. Richmond et al., EMBO J. 7 [1988] 2025-2033) the sequence of which closely resembles that deduced from the gro-cDNA isolated from fibroblasts (A. Anisowicz et al., Proc. Nat. Acad. USA 84 [1987] 7188-7192). A murine macrophage-derived inflammatory protein (MIP), possibly related to the proteins described above, was also reported (G. Davatelis et al., J. Exp. Med. 167 [1988] 1939-1944) and appears to be a member of a family of peptides including RANTES, the 8 kd product of a cDNA isolated from IL-2 dependent, antigen-driven human T-cell clones (T. J. Schall et al., J. Immunol. 141 [1988] 1018-1029). The functions of these peptides are largely unknown. MGSA and CTAP-III (C. W. Castor et al., Proc. Nat. Acad. Sci. USA 80 [1983] 765-769) were reported to be mitogenic, and platelet factor 4 was shown to have immunomodulatory effects (A. D. Barone et al., J. Biol. Chem. 263 [1988] 8710-8715).
The results obtained suggest that NAF selectively activates neutrophils by a receptor-mediated process similar to that initiated by chemotactic agonists.
The NAF of this invention was found to induce oxygen radical formation and enzyme release in human neutrophils by acting via a selective receptor, different from all receptors thus far described.
The total amino acid sequence of NAF purified from LPS-stimulated human monocytes has been determined and a gene coding for the main NAF species synthesized and expressed as a recombinant peptide with the same neutrophil-activating properties as its natural homologue.
The invention thus concerns NAF and its isolation from natural sources such as human monocytes.
It also concerns synthetic, especially recombinant NAF and its preparation and expression.
The complete amino acid sequence of NAF was determined by known sequencing methods:
Ser-Ala-Lys-Glu-Leu-Arg-Cys-Gln-Cys-Ile-Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His-Pro-Lys-Phe-Ile-Lys-Glu-Leu-Arg-Val-Ile-Glu-Ser-Gly-Pro-His-Cys-Ala-Asn-Thr-Glu-Ile-Ile-Val-Lys-Leu-Ser-Asp-Gly-Arg-Glu-Leu-Cys-Leu-Asp-Pro-Lys-Glu-Asn-Trp-Val-Gln-Arg-Val-Val-Glu-Lys-Phe-Leu-Lys-Arg-Ala-Glu-Asn-Ser.
Edman degradation of the full protein and of typtic fragments T 7-26 and T 27-47 obtained after splitting of SH-methylated and amino-succinylated NAF gave the sequence up to position 47. Upon hydrolysis with 75% formic acid and Edman degradation two approximately equimolar sequences of 20 amino acids were obtained, followed by a single pattern corresponding to the aminoterminal sequence of NAF beyond position 20. The sequence of the carboxyterminal peptide A 53-72 was determined by subtraction and confirmed by the analysis of the typtic peptide T 61-72. Carboxypeptidase- A and -B give no detectable cleavage product, but after treatment of 1 nmol NAF with carboxypeptidase Y 120 pmol serine are released, indicative of serine as the carboxyterminus.
Analysis of various batches of NAF revealed some aminoterminal heterogeneity. The above sequence is for the major component (about 70%), which is believed to be largely responsible for the biological activities. Three further variants could be identified:
Sequence 1 (about 17%):
Ala-Val-Leu-Pro-Arg-Ser-Ala-Lys-Glu-Leu-Arg-Cys-Gln-Cys-Ile-Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His-Pro-Lys-Phe-Ile-Lys-Glu-Leu-Arg-Val-Ile-Glu-Ser-Gly-Pro-
i.e the above full sequence further extended at the N-terminus by Ala-Val-Leu-Pro-Arg- (the full protein thus has 77 amino acids)
Sequence 2 (about 8%):
Lys-Glu-Leu-Arg-Cys-Gln-Cys-Ile-Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His-Pro-Lys-Phe-Ile-Lys-Glu-Leu-Arg-Val-Ile-Glu-Ser-Gly-Pro-
i.e. the above full sequence shortened at the N-terminus by Ser-Ala- (the full protein thus has 70 amino acids)
Sequence 3 (about 5%):
Glu-Leu-Arg-Cys-Gln-Cys-Ile-Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His-Pro-Lys-Phe-Ile-Lys-Glu-Leu-Arg-Val-Ile-Glu-Ser-Gly-Pro-.
i.e. the above full sequence shortened at the N-terminus by Ser-Ala-Lys- (the full protein thus has 69 amino acids).
All these sequences can be aligned to the predicted sequence from the 3-10C cDNA, consisting of 99 amino acids (J. Schmid and C. Weissmann, J. Immunol. 139 [1987] 250-254). The major NAF peptide corresponds to positions 28 to 99 of the 3-10C amino acid sequence, while the three above variants start at positions 23 (sequence 1), 30 (sequence 2) and 31 (sequence 3) thereof.
Comparison of the 3-10C cDNA-derived sequence with the above sequence data suggests that mononuclear phagocytes synthesize a precursor of NAF which is processed intracellularly. The 77-residue peptide could represent the largest secreted form of NAF as it corresponds to the cDNA-derived sequence minus the predicted signal peptide of 22 amino acids. The main, 72-residue NAF species and the analogues with 70 and 69 residues may arise from further posttranslational modifications.
All forms of NAF have four cysteine residues expected to form intrachain disulfide bridges which appear important for activity since mercaptoethanol is found to be inhibitory (P. Peveri et al., J. Exp. Med. 167 [1988] 1547-1560). The disulfide bridges are likely to link the positions 7-34 and 9-50 as in beta-thromboglobulin and platelet factor 4 which are partially homologous to NAF.
In accordance with the homology to xcex2-thromboglobulin NAF has two intramolecular disulfide bridges. Consequently the calculated molecular weight is 8384.7, clearly higher than the apparent molecular weight found by SDS-gel electrophoresis.
The production of NAF from natural sources, such as human monocytes, results in poor yields and requires extensive and complicated purification steps.
In order to produce NAF in larger amounts and better yield synthetic processes, e.g. total chemical synthesis or recombinant DNA processes, are indicated. The production of synthetic NAF, e.g. by recombinant DNA methods, including expression in a procaryotic or eucaryotic expression system, e.g. in E. coli are also part of the invention. Synthetic NAF, i.e. NAF produced by synthetic processes such as recombinant DNA processes, is also part of the invention. Synthetic NAF, e.g. the recombinant NAF isolated e.g. from bacteria has the same amino acid sequence(s) as natural NAF and the same biological properties and activities. xe2x80x9cRecombinant NAFxe2x80x9d means NAF obtained by recombinant DNA processes.
The preparation of NAF by recombinant DNA methods is effected according to known procedures, namely synthesis, purification and ligation of corresponding oligonucleotides, cloning of the synthetic NAF gene, expression in e.g. E. coli and finally recovery and purification of recombinant NAF. For example, a gene coding for the 72-amino acid NAF is synthesized, preferably with codon optimization, then cloned and expressed in E. coli. 
Western blot analysis of crude bacterial extracts using an antiserum raised against natural NAF reveals a single band that comigrates with natural NAF. Recombinant NAF purified to homogeneity has identical amino- and carboxyterminal sequences as the 72-amino acid natural NAF. If tested on human neutrophils it is found to have the same activity and potency as natural NAF in inducing chemotaxis, a rapid rise in cytosolic free Ca2+, activation of the respiratory burst, and release of specific and azurophil granule contents.
FIG. 13 shows the design of a synthetic NAF gene. Changes are apparent over the coding sequence of the 3-10C cDNA to facilitate expression in E. coli. At the 3xe2x80x2-end, the naturally-occurring terminator TAA is replaced by a triple terminator TAATAATGA, and a BamHI site is added immediately downstream. At the 5xe2x80x2-end, SphI and ClaI sites are added to allow insertion into the cloning and the expression vector, respectively. At base 34, a TaqI restriction site is created for later 5xe2x80x2-end manipulations by changing the Arg codon from AGA to CGA.
Blockwise annealing (oligonucleotides 1-2, 3-4 and 5-6) has no advantage over the simultaneous ligation of all six oligonucleotides. 5xe2x80x2-Phosphorylation of all oligonucleotides gives rise to ligation products larger than the expected size, but when the terminal oligonucleotides (1 and 6) are not phosphorylated, the product with the highest molecular weight is the complete gene with 248/240 bases. The gene is ligated into SphI/BamHI-cut pBS M13xe2x88x92 and transformed into E. coli. Plasmid DNA isolated from 6 of the resulting ampicillin-resistant colonies is cut with ClaI/BamHI, yielding in all cases a 237 bp band on a 1.4% agarose gel. DNA sequencing shows the clones containing the correct sequence. The ClaI/BamHI band from a correct clone is removed from the gel and cloned into the Trp promoter vector pIL402(Term). As a possible means of increasing expression, a synthetic transcription terminator (see Example 17) may be incorporated into this vector attached to the 3xe2x80x2-end of the human GM-CSF gene at the BamHI site. The NAF gene is therefore inserted between this BamHI site and the ClaI site downstream of the transcription initiation site in the promotor, to replace the GM-CSF gene. The resulting NAF expression plasmid, p(NAF)-6T3, is shown in FIG. 14.
Silver stain and western blot analysis of extracts of indole acrylic acid-induced E. coli containing p(NAF)-6T3 demonstrates the time-dependent production of a peptide that comigrates with natural NAF and reacts with an antiserum against natural NAF.
The above expression system can also be used to prepare the NAF variants mentioned above or other biologically active NAF fragments such as amino-truncated NAF fragments.
The biological properties of NAF are species-specific. Activity in the rabbit most closely mirrors activity in humans. Preliminary tests in the mouse, rat and guinea pig have not shown any clear activity.
NAF is indicated for use in the treatment of conditions which are accompanied or caused, locally or systemically, by a modification of the number or activation state of the PMN (polymorphonuclear cellsxe2x80x94neutrophils). NAF extensively modifies these PMN parameters and is therefore indicated for use in the treatment of conditions in which an elevation of the number or activation state of the PMN leads to clinical improvement, e.g. in bacterial, mycoplasma, yeast, and fungal, in and in viral infections. Furthermore NAF is indicated for use in inflammatory illnesses such as psoriasis, arthritic conditions and asthma, or in conditions of abnormally low neutrophil count and/or generalized low neutrophil level, and in the preparation of antagonists for use in these indications.